Primorigen Biosciences will use SBIR funds to develop a frameless, multiplexed, microarray screening system for rapid identification and characterization of antibody matched pairs with appropriate affinity and specificity for human proteins central to pluripotency and multipotency. This system produces improved assay content and dramatically reduces the time and cost of multiplexed microarray development. The core of this new system is Primorigen's novel frameless microarray device that allows multiplexed analysis of 96 (or 384) samples against several binding targets directly on a flat array surface. As a result, dozens of proteins in hundreds of samples can be detected with high precision to generate thousands of data points per experiment, increasing speed of screening. While the new system can be adapted for detection of any protein, this Phase I SBIR project will focus on characterizing assay content for detecting eight human cell markers of pluripotency and multipotency and validating them in a multiplexed array format. Phase II will a) complete additional microarrays of up to 15-20 multiplex assays, b) further develop automated methods to increase assay throughput, c) apply this system to pathway analysis involved in determining or maintaining cell potency and d) expand assay development to endoderm, mesoderm, and ectoderm markers. PUBLIC HEALTH RELEVANCE: Proteins facilitate many of the cell's most basic functions and thousands of new protein sequences and post-translational variants have been identified in the past few years. This protein jackpot has hastened the drive to understand protein-based regulation, but researchers require better biophysical methods to quantitatively detect protein markers. Primorigen's inexpensive, miniaturized, multiplexed, high throughput assays proposed for SBIR support will address this need, generating thousands of protein detection data points per experiment. At the same time, the project will provide new antibody and protein content for assays to detect human proteins central to pluripotency and multipotency.